Glomeruli are the structures around which neurons are organized in the olfactory bulb. Each glomerulus carries information about a single odorant receptor in the nose, and has a specific population of glutamatergic mitral cells (MCs) and tufted cells (TCs) associated with it. The broad objective of this proposal is to build on provocative recent results from a variety of labs, including our own, pertaining to the cellular connectivity within a glomerulus. These studies suggest that most signaling between olfactory sensory neurons (OSNs) and MCs occurs through a multi-step path involving intermediary glutamatergic TCs (OSN-to-TC-to-MC), differing from the canonical model of direct OSN-to-MC excitation. In addition, glutamatergic signals from TCs are unusual, being mediated by long-range extrasynaptic transmission. These results are however controversial, as limited ultrastructural data suggest that OSNs are at least capable of making direct morphological contacts on MCs. In this application, we propose in Aim 1 to apply new approaches, including quantitative ultrastructural analyses, to establish whether the multi-step path is indeed the dominant signaling mechanism between OSNs and MCs. Aims 2 and 3 will then examine possible functional implications of a multi-step path for MCs using electrophysiological recordings in brain slices and awake behaving mice. The extrasynaptic nature of glutamate signaling from TCs, in particular, predicts that excitation of MCs should be highly non-linear, reflecting the accumulation of glutamate. For odor responses, this could, mean, for example, that MC responses to different odors are exceptionally sparse and display a steep dependence on odor concentration. A key tool in our functional studies will be connexin-36 knockout mice. Because OSN signaling onto MCs in these mice appears to be converted from multi-step to monosynaptic, they provide a potentially direct way to assess whether the multi-step activation path for MCs is causally related to specific functional properties. Taken together, our studies wil lead to important new information about the relationship between the structure and function of an olfactory circuit.